We propose to continue our studies of the initiation of bacterlophage lambda DNA replication in a system that is reconstituted with highly purified lambda0 and E. coli proteins. Our long range goal is to achieve a detailed mechanistic understanding of the biochemical events that occur in the initiation, propagation and regulation of lambda DNA replication. We will perform mechanistic studies of a ten protein system that specifically initiates DNA replication at a lambda replication origin (ori lambda) present on a superconed plasmid template. We will attempt to establish bidirectional replication in this system. We will conduct high-resolution supercoil footprinting analysis of the various nucleoprotein prepriming structures that are formed at ori lambda prior to the initiation of localized DNA unwinding, priming and DNA chain synthesis. The effect of template superhelicity on the efficiency of initiation of lambda replication will be determined. We will examine how E. coli HU protein, a strong inhibitor of lambda DNA replication, blocks the initiation process. The molecular mechanisms involved in the transcriptional activation of lambda DNA replication will be thoroughly explored. We will mutagenize the A/T-rich region of ori lambda and select for origin defective mutants to determine which nucleotide residues play a critical role in the initiation of chromosomal DNA replication. We will continue our studies of the propagation of lambda replication forks on rolling-cycle DNA templates. In particular, we will investigate how highly processive replication forks deal with stable nucleoprotein structures that they encounter during their rapid movement along the chromosome. We will use immunoelectron microscopy to determine which replication proteins are directly associated with the replication fork on a rollingcycle template. Much of our efforts during the next project period will be devoted to studies of the lambda O initiator protein. These studies will include (a) a determination of the binding constant for interaction of O with its recognition site; (b) quantitative analysis of the individual-site binding isotherms for O binding to ori lambda, using footprinting and isothermal titration calorimetry; (c) formation and analysis of cocrystals of O protein with its DNA binding site; (d) isolation and characterization of DNA-binding mutants in O protein; (e) isolation and characterization of mutants in the O protein recognition site; and (f) purification and characterization of the carboxy-terminal domain of O protein. Biochemical studies of lambda DNA replication have provided and will undoubtedly continue to provlde important insights into the biological mechanisms used in the initiation and regulation of chromosomal DNA replication. This knowledge, furthermore, provides important guidelines for studies of these processes in more complex eukaryotic systems.